Downy mildew resistant lettuce plants

ABSTRACT

The present disclosure provides lettuce plants exhibiting resistance to downy mildew. Such plants may comprise novel introgressed genomic regions associated with disease resistance from  L. virosa . In certain aspects, compositions, including novel polymorphic markers and methods for producing, breeding, identifying, and selecting plants or germplasm with a disease resistance phenotype are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority of U.S. Provisional Appl. Ser. No.62/214,097, filed Sep. 3, 2015, the entire disclosure of which isincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to the field of agriculture and morespecifically to methods and compositions for producing lettuce plantsexhibiting improved disease resistance.

INCORPORATION OF SEQUENCE LISTING

The sequence listing that is contained in the file named“SEMB018US_ST25.txt”, which is 6.83 kilobytes as measured in MicrosoftWindows operating system and was created on Aug. 31, 2016, is filedelectronically herewith and incorporated herein by reference.

BACKGROUND OF THE INVENTION

Disease resistance is an important trait in agriculture, particularlyfor the production of food crops. Although disease resistance alleleshave been identified in wild lettuce lines, efforts to introduce thesealleles into cultivated lines are hindered by a lack of specific markerslinked to the alleles, linkage drag that leads to unacceptable plantquality and a lack of durable resistance. The use of marker-assistedselection (MAS) in plant breeding methods has made it possible to selectplants based on genetic markers linked to traits of interest. However,accurate markers for identifying or tracking desirable traits in plantsare frequently unavailable even if a gene associated with the trait hasbeen characterized. These difficulties are further complicated byfactors such as polygenic or quantitative inheritance, and an oftenincomplete understanding of the genetic background underlying expressionof a desired phenotype.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides an elite lettuce plantcomprising an introgression from Lactuca virosa on chromosome 7, whereinsaid introgression comprises a single gene conferring improvedresistance to Bremia lactucae relative to a plant lacking saidintrogression.

In another aspect, the present invention provides an elite lettuce plantcomprising an introgression from Lactuca virosa on chromosome 7, whereinsaid introgression comprises a first allele conferring improvedresistance to Bremia lactucae relative to a plant lacking saidintrogression, and wherein said introgression lacks a second allelegenetically linked to said first allele and conferring stunted growth.In some embodiments, the introgression from Lactuca virosa is locatedbetween 53.6 Mb and 55.2 Mb. In other embodiments, the introgressioncomprises a first allele at position 53,628,734 bp conferring improvedresistance to Bremia lactucae relative to a plant lacking said firstallele, and wherein said introgression lacks a second allele geneticallylinked to said first allele at position 55,223,518 bp. In furtherembodiments, the introgression from Lactuca virosa is about 250 kb orless. The invention further provides an elite lettuce plant comprising aLactuca virosa allele at locus NLSAT009419770, and wherein the plantlacks a Lactuca virosa allele at locus ND0229340 or NLSAT005132975. Inspecific embodiments, the plant comprises a Lactuca virosa allele atlocus NLSAT009419770 (SEQ ID NO: 6) and a Lactuca sativa allele at locusNLSAT005132975 (SEQ ID NO:1), or is defined as comprising a Lactucavirosa allele at locus NLSAT009419770 (SEQ ID NO: 6) and a Lactucasativa allele at locus ND0229340 (SEQ ID NO: 11). In certain additionalembodiments, the invention provides an introgression, wherein a sampleof seed comprising the introgression was deposited under ATCC AccessionNumber PTA-121598. In further embodiments, the invention provides aplant part of a lettuce plant of the invention, wherein the plant partis a cell, a seed, a root, a stem, a leaf, a head, a flower, or pollen.

In a further aspect, the invention provides a method for producing alettuce plant with improved resistance to Bremia lactucae, comprising:a) crossing an elite lettuce plant of the invention with itself or witha second lettuce plant of a different genotype to produce one or moreprogeny plants; and b) selecting a progeny plant comprising saidintrogression. In some embodiments, selecting the progeny plantcomprises identifying a progeny plant that (1) comprises a Lactucavirosa allele at a locus genetically linked to said first allele and/orlacks an elite allele present at the corresponding locus in the elitelettuce plant, and (2) lacks a Lactuca virosa allele at a locusgenetically linked to said second allele, and/or comprises an allelepresent at the corresponding locus from in the elite lettuce plant. Infurther embodiments, selecting a progeny plant comprises detecting atleast one allele at a locus selected from the group consisting ofNLSAT009419770, ND0229340 and NLSAT005132975. In certain embodiments,the allele(s) are detected by a PCR-based method using oligonucleotideprimer pair(s); and the allele at locus NLSAT009419770 is detected usingthe primer pair comprising SEQ ID NO: 7 and SEQ ID NO: 8; or the alleleat locus ND0229340 is detected using the primer pair comprising SEQ IDNO: 12 and SEQ ID NO: 13; or the allele at locus NLSAT005132975 isdetected using the primer pair comprising SEQ ID NO: 2 and SEQ ID NO:3.In certain other embodiments, selecting a progeny plant comprisesdetecting an allele at locus NLSAT009419770 and an allele at locusND0229340. The progeny plant may be an F2-F6 progeny plant. Producingthe progeny plant may comprise backcrossing, for example between 2-7generations of backcrosses. In further embodiments, a sample of seedcomprising said introgression was deposited under ATCC Accession NumberPTA-121598.

In yet another aspect, the invention provides a method for obtaining anelite lettuce plant exhibiting improved resistance to Bremia lactucae,comprising: a) obtaining an elite lettuce plant heterozygous for a firstallele from Lactuca virosa that confers quantitative resistance toBremia lactucae and that is genetically linked in the plant to a secondallele from Lactuca virosa that confers stunted growth, wherein theplant is heterozygous relative to a corresponding locus in said eliteplant; (b) obtaining progeny of the plant; and (c) selecting at least afirst progeny plant in which recombination has occurred such that theprogeny comprises said first allele that confers quantitative resistanceto Bremia lactucae but not said second allele. In some embodiments,selecting the progeny plant comprises identifying a progeny plant thatcomprises a Lactuca virosa allele at a locus genetically linked to saidfirst allele and/or lacks an allele present at the corresponding locusin the elite lettuce plant, and lacks a Lactuca virosa allele at a locusgenetically linked to said second allele, and/or comprises an allelepresent at the corresponding locus from in the elite lettuce plant. Inother embodiments, selecting a progeny plant comprises detecting atleast one allele at a locus selected from the group consisting ofNLSAT009419770, ND0229340 and NLSAT005132975. In further embodiments,(a) the allele(s) are detected by a PCR-based method usingoligonucleotide primer pair(s); and (b) the allele at locusNLSAT009419770 is detected using the primer pair comprising SEQ ID NO: 7and SEQ ID NO: 8; or the allele at locus ND0229340 is detected using theprimer pair comprising SEQ ID NO: 12 and SEQ ID NO: 13; or the allele atlocus NLSAT005132975 is detected using the primer pair comprising SEQ IDNO: 2 and SEQ ID NO:3. In certain embodiments, selecting a progeny plantcomprises detecting an allele at locus NLSAT009419770 and an allele atlocus ND0229340. Further embodiments provide a plant produced by themethods of the invention, for example a cell, a seed, a root, a stem, aleaf, a head, a flower, and pollen.

In another aspect, the invention provides an elite lettuce plant,wherein a sample of seed of said plant was deposited under ATCCAccession Number PTA-121598.

In yet another aspect, the invention provides a method for producing alettuce plant with improved resistance to Bremia lactucae, comprising:a) crossing an elite lettuce plant wherein a sample of seed of saidplant was deposited under ATCC Accession Number PTA-121598 with itselfor with a second lettuce plant of a different genotype to produce one ormore progeny plants; and b) selecting a progeny plant comprising anintrogression from Lactuca virosa on chromosome 7, wherein saidintrogression comprises a first allele conferring improved resistance toBremia lactucae relative to a plant lacking said first allele, andwherein said introgression lacks a second allele genetically linked tosaid first allele and conferring stunted growth. In certain embodiments,selecting a progeny plant comprises detecting at least one allele at alocus selected from the group consisting of NLSAT009419770, ND0229340and NLSAT005132975. In other embodiments, (a) the allele(s) are detectedby a PCR-based method using oligonucleotide primer pair(s); and (b) theallele at locus NLSAT009419770 is detected using the primer paircomprising SEQ ID NO: 7 and SEQ ID NO: 8; or the allele at locusND0229340 is detected using the primer pair comprising SEQ ID NO: 12 andSEQ ID NO: 13; or the allele at locus NLSAT005132975 is detected usingthe primer pair comprising SEQ ID NO: 2 and SEQ ID NO:3. In furtherembodiments, selecting a progeny plant comprises detecting an allele atlocus NLSAT009419770 and an allele at locus ND0229340. The inventionfurther provides a plant produced by the methods of the invention.

In certain embodiments, the invention provides a marker for identifyinga plant with improved resistance to Bremia lactucae selected from thegroup consisting of NLSAT005132975 (SEQ ID NO:1), NLSAT009419770 (SEQ IDNO: 6), and ND0229340 (SEQ ID NO: 11). In one embodiment, the markercomprises NLSAT005132975 (SEQ ID NO:1), or NLSAT009419770 (SEQ ID NO:6), or ND0229340 (SEQ ID NO: 11). In other embodiments, more than one orall three markers may be used. In further embodiments, a marker linkedto or associated with any of the markers described herein may be usefulin accordance with the invention. For example, such a marker may belocated within 40 cM, 20 cM, 15 cM, 10 cM, 5 cM, 2 cM, or 1 cM of amarker associated with disease resistance described herein. In otherembodiments, a marker in accordance with the invention may comprise 100%sequence identity or homology to a marker described herein, or maycomprise 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50% sequenceidentity or homology to a marker described herein. In still furtherembodiments, a marker useful with the invention may be comprised withina plant genome, such as a lettuce plant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and FIG. 1B: Shows a physical map of a Lactuca virosaintrogression segment and the positions of markers used forrecombination screenings.

FIG. 2: Shows marker sequences NLSAT009419770 (SEQ ID NO: 6), ND0229340(SEQ ID NO: 11) and NLSAT005132975 (SEQ ID NO:1). Polymorphicnucleotides between L. virosa and L. sativa are indicated.

DETAILED DESCRIPTION

Downy mildew (DM) is a damaging fungal disease caused by Bremialactucae, which can result in severe crop loss in lettuce plants(Lactuca sativa). Several wild lettuce species, including Lactucaserriola and Lactuca virosa, are known to exhibit resistance to DM, andintensive efforts have been made to introgress DM resistance allelesfrom these species into cultivated lettuce types. However, these effortshave been unsuccessful in producing commercially acceptable lettuce withresistance to DM. The previously known alleles have failed to providereproducible results, provide durable DM resistance or have beenaccompanied by undesirable agronomic traits. Downy mildew remains aleading cause of crop loss in lettuce.

The present invention provides novel introgressions of diseaseresistance alleles from Lactuca virosa. These alleles can beintrogressed into cultivated lettuce lines, resulting in lettuce plantswhich exhibit DM resistance. In addition, these novel introgressions aresmaller fragments from the wild species and therefore also have areduced risk of linked, deleterious traits or linkage drag. Theinvention represents a significant advance in the art. The inventionfurther provides novel markers for tracking the genomic segments. Thenovel fragment can be used to introgress DM disease resistance into anydesired lettuce genotype.

Resistance to downy mildew has been obtained with introgressions fromthe wild lettuce Lactuca serriola, which can be successfully crossedwith cultivated lettuce types and generally does not have significantlinkage drag. However, the B. lactucae pathogen is highly geneticallyvariable, and has rapidly adapted to the resistance alleles which havebeen found in L. serriola. As such, there is an ongoing need for theidentification of new resistance alleles to target the new isolates ofthe pathogen. The development of new alleles is costly and laborious. Todate, L. serriola has not provided durable DM resistance.

The wild lettuce L. virosa has provided a durable source of resistanceto Nasonovia ribisnigri race 0 that has lasted for many years. However,to date, the resistance alleles to DM from L. virosa which have beendescribed have not succeeded in providing durable resistance. Inaddition, the DM resistance alleles from L. virosa have required thepresence of two or more genes, have resulted in significant linkage dragand/or have not been able to be successfully reproduced or successfullyincorporated into cultivated lettuce for resistance. L. virosa is alsodifficult to cross with cultivated lettuce lines and the progeny plantsgenerally exhibit severe linkage drag effects as a result of thepresence of an L. virosa introgression.

Prior efforts to introgress DM resistance alleles from L. virosa havenot been successful in providing commercially acceptable lettuce plantswith resistance to DM. The prior efforts have not been reproducible,have provided relatively large introgressions with significant linkagedrag and/or have required the presence of multiple genes. The largerintrogressions from L. virosa exacerbate the deleterious traitstypically observed with L. virosa. Introgression of any alleged L.virosa DM resistance alleles has also been complicated by a lack ofmarkers and assays that accurately correlate genotype with resistanceover a variety of lettuce lines. The use of markers in developinglettuce lines with resistance to DM from L. virosa has been hindered bya lack of predictive markers. The previously reported markers foridentifying or tracking DM resistance in lettuce plants could not bereproduced. Moreover, previously identified markers are not predictiveacross multiple plant lines, or are not tightly linked to DM resistanceloci and are therefore ineffective when disease resistance is crossedinto a related species. For example, the use of random amplifiedpolymorphic DNA (RAPD) assays for the identification and tracking of DMresistance and lettuce plants has been found not to be reproducible(Jones, et al., 1997, Molecular Breeding 3:381-390), and previouslyreported RAPD markers are not reliably useful in detecting a givenresistance gene in different plant populations (Kelly, et al., 1995,HortScience 30:461-465).

Despite the many obstacles to the successful introgression of L. virosaresistance alleles into cultivated lettuce lines, the present inventorswere able to produce novel introgressions from L. virosa which confer DMresistance. These novel introgressions are smaller and lack or reducethe deleterious traits previously associated with L. virosa crosses. Inaddition, the present invention includes novel trait-linked markerswhich can be used to make novel recombined introgressions on chromosome7 and confer DM resistance. The invention further identifies andprovides a genomic segment from approximately 53.4 Mb to 56.1 Mb onchromosome 7 associated with DM resistance. The present inventionfurther provides a novel introgression fragment from L. virosa having asize of about 250 kb or less. Surprisingly, the novel introgressionresults in plants exhibiting DM resistance. In addition, certain linkagedrag often associated with introgressions from L. virosa, includingstunted growth, has not been observed with the novel introgressionprovided herein. The novel introgression provided by the inventiontherefore yields lettuce plants resistant to DM while beneficiallyreducing or eliminating the introduction of negative agronomic traitsfrom L. virosa into an elite background.

The invention further provides novel markers and assays that allow theaccurate identification and tracking of the genomic regions providedherein. Because genetically diverse lettuce lines can be difficult tocross due in part to suppressed recombination, the introduction of DMresistance alleles from L. virosa into elite lines using conventionalbreeding methods would require prohibitively large segregatingpopulations for progeny screens with an uncertain outcome.Marker-assisted selection (MAS) is therefore important for the effectiveintrogression of wild lettuce alleles into elite cultivars. However,previously known markers for DM resistance have been shown to benon-reproducible or inaccurate, perhaps because of poor quality of themarkers or a lack of understanding of the mechanisms controlling DMresistance and the inability to resolve the specific regions associatedwith resistance. In contrast, the present invention allows for MAS byproviding improved and validated markers for detecting genotypesassociated with disease resistance.

II. Genomic Regions, Alleles, and Polymorphisms Associated With DownyMildew Resistance in Lettuce Plants

DM can infect lettuce plants at any stage in the growth cycle and cancause severe reduction in yield and quality in a lettuce crop. Cultivarresistance is the most economically feasible way of controlling DMinfection due the high cost of fungicide spray applications. Intensiveefforts have therefore been made to identify effective sources of DMresistance. However, previously known introgressions from wild specieshave not produced commercially useful lettuce crops due to a lack ofdurable resistance or unacceptable associated deleterious traits.

Other accessions of L. virosa exhibiting DM resistance are known in theart and may be used in accordance with certain embodiments of theinvention. It may also be possible to use other wild lettuce typesincluding L. serriola, and L. saligna. Accessions for L. serriola linesexhibiting resistance to various B. lactucae races are given, forexample, in Lebeda, et al., Eur J Plant Pathol, 138:597-640, 2014.Lactuca saligna accession CGN05271, which can be obtained from theCenter for Genetic Resources (Wageningen, The Netherlands) also exhibitsDM resistance. L. virosa exhibiting DM resistance is described inLebeda, et al., 2014. L. virosa accessions have been collected innumerous locales including France and Portugal and can be found in anumber of germplasm banks including Center for Genetic Resources, theNetherlands (CGN) and National Plant Germplasm System (NPGS).

Other DM resistance sources have also been described and are known inthe art (see, for example, Lebeda, et al., Molecular Plant Pathology,44:125-139, 1995; Bonnier, et al., Euphytica 61: 203-211, 1992;Gustafsson, Euphytica, 40: 227-232, 1989; and Netzer, Hort. Sci.,11:612-613, 1976).

Efforts to introgress DM resistance into cultivated lettuce lines havealso been unsuccessful or complicated by the fact that previouslydescribed introgressions have been very large or are derived fromintrogressions occurring at several distinct loci. In addition, previousstudies have reported that two or more resistance genes from L. virosaare required for durable DM resistance in lettuce. However, theintroduction of a large amount of genetic material by introgressinglarge genomic segments greatly increases the incidence of negativetraits as a result of linkage drag. Despite numerous efforts, no allelesfrom L. virosa which confer durable resistance to DM have yet beendiscovered.

A further hindrance to the effective identification of DM resistancealleles and introgression into cultivated lettuce lines is the highgenetic variability of the B. lactucae pathogen. The geneticadaptability of the pathogen allows it to overcome resistance derivedfrom certain species of wild lettuce. For example, resistance allelesfrom L. serriola have been shown to be readily “broken down” by B.lactucae. Moreover, known B. lactucae resistance alleles have been shownto have varying resistance to different B. lactucae races.

It is therefore desirable to identify smaller genomic regions conferringDM resistance. It is also desirable to provide resistance with a singlegene and to provide a gene having more durable resistance. Such smallerregion may not include alleles associated with linkage drag. Inaddition, resistance residing in smaller introgressions or associatedwith a single gene is also less likely to be broken down duringbreeding, which results in more durable resistance. The introgressionidentified herein or any introgression with a reduced introgression sizehas not been achieved previously. Moreover, no effective markers whichare closely linked to resistance have been described.

Using the genetic markers and assays of the invention, Applicants wereable to successfully identify a novel DM resistance region from L.virosa. This region also results in a reduction or elimination oflinkage drag associated with L. virosa. In certain embodiments, thenovel introgression of the present invention spans a region defined asbetween about 49.8 cM and 50.5 cM on Linkage Group 7. Public markersthat define Linkage Group 7 have been published by McHale (McHale etal., Theor Appl Genet (2009) 118:565-580) and Truco (Truco et al., 2013G3 (2013) 3: 617-631). The genetic interval appeared to correspond tothe physical interval of approximately 53.4 Mb to 56.1 Mb as determinedbased on a genome consensus map generated in a collaboration of theMichelmore lab at UC Davis and the BGI, Shenzen employing genomesequence data from the Lettuce Genome Sequencing Consortium (availableat lgr.genomecenter.ucdavis.edu/Home.php). One of skill in the art willunderstand that interval values may vary based on factors such as thereference map that is used, the sequencing coverage and the assemblysoftware settings. However, such parameters and mapping protocols areknown in the art and one of skill in the art can use the markersequences provided herein to physically and genetically anchor theintrogressions described herein to any given map using such methodology.

The invention further provides a novel introgression from L. virosawhich is much smaller than previously known introgressions, and whicheffectively confers resistance in an elite background. In addition, thesmaller introgression reduces negative linkage drag. In someembodiments, the DM resistance locus has an introgression size of 250 kbor less. The novel introgression of the present invention thereforeconfers significantly improved agronomic properties over previouslettuce lines.

III. Introgression of Genomic Regions Associated with Disease Resistance

Marker-assisted introgression involves the transfer of a chromosomalregion defined by one or more markers from a first genetic background toa second. Offspring of a cross that contain the introgressed genomicregion can be identified by the combination of markers characteristic ofthe desired introgressed genomic region from a first genetic backgroundand both linked and unlinked markers characteristic of the secondgenetic background.

The present invention provides novel accurate markers for identifyingand tracking introgression of one or more of the genomic regions from L.virosa disclosed herein into cultivated lines. The invention furtherprovides markers for identifying and tracking the novel introgressionsdisclosed herein during plant breeding, including markers NLSAT005132975(SEQ ID NO:1), NLSAT009419770 (SEQ ID NO: 6), and ND0229340 (SEQ ID NO:11).

Markers within or linked to any of the genomic intervals of the presentinvention may be useful in a variety of breeding efforts that includeintrogression of genomic regions associated with disease resistance intoa desired genetic background. For example, a marker within 40 cM, 20 cM,15 cM, 10 cM, 5 cM, 2 cM, or 1 cM of a marker associated with diseaseresistance described herein can be used for marker-assistedintrogression of genomic regions associated with a disease tolerantphenotype.

Lettuce plants comprising one or more introgressed regions associatedwith a desired phenotype wherein at least 10%, 25%, 50%, 75%, 90%, or99% of the remaining genomic sequences carry markers characteristic ofthe germplasm are also provided. Lettuce plants comprising anintrogressed region comprising regions closely linked to or adjacent tothe genomic regions and markers provided herein and associated with adisease resistance phenotype are also provided.

IV. Development of Disease Resistant Lettuce Varieties

For most breeding objectives, commercial breeders work within germplasmthat is “cultivated type” or “elite.” This germplasm is easier to breedbecause it generally performs well when evaluated for horticulturalperformance. A number of cultivated lettuce types have been developed,including L. sativa, which is agronomically elite and appropriate forcommercial cultivation. Lettuce cultivar groups include, but are notlimited to the Cos, Cutting, Stalk (or Asparagus), Butterhead, Crisphead(or Iceberg or Cabbage), Latin and Oilseed groups (De Vries, Gen.Resources and Crop Evol. 44: 165-174, 1997). However, the performanceadvantage a cultivated germplasm provides can be offset by a lack ofallelic diversity. Breeders generally accept this tradeoff becauseprogress is faster when working with cultivated material than whenbreeding with genetically diverse sources.

When cultivated germplasm is crossed with non-cultivated germplasm, abreeder can gain access to novel alleles from the non-cultivated type.However, this approach presents significant difficulties due tofertility problems associated with crosses between diverse lines, andnegative linkage drag from the non-cultivated parent. In lettuce plants,non-cultivated types such as L. serriola or L. virosa can providealleles associated with disease resistance. However, thesenon-cultivated types may have poor horticultural qualities.

The process of introgres sing desirable resistance genes fromnon-cultivated lines into elite cultivated lines while avoiding problemswith linkage drag or low heritability is a long and often arduousprocess. Success in deploying alleles derived from wild relativestherefore strongly depends on minimal or truncated introgressions thatlack detrimental effects and reliable marker assays that replacephenotypic screens. Success is further defined by simplifying geneticsfor key attributes to allow focus on genetic gain for quantitativetraits such as disease resistance. Moreover, the process ofintrogressing genomic regions from non-cultivated lines can be greatlyfacilitated by the availability of accurate markers for MAS.

One of skill in the art would therefore understand that the alleles,polymorphisms, and markers provided by the invention allow the trackingand introduction of any of the genomic regions identified herein intoany genetic background. In addition, the genomic regions associated withdisease resistance disclosed herein can be introgressed from onegenotype to another and tracked using MAS. Thus, Applicants' discoveryof accurate markers associated with disease resistance will facilitatethe development of lettuce plants having beneficial phenotypes. Forexample, seed can be genotyped using the markers of the presentinvention in order to select for plants comprising desired genomicregions associated with disease resistance. Moreover, MAS allowsidentification of plants which are homozygous or heterozygous for adesired introgression.

Inter-species crosses can also result in suppressed recombination andplants with low fertility or fecundity. For example, suppressedrecombination has been observed for the tomato nematode resistance geneMi, the Mla and Mlg genes in barley, the Yr17 and Lr20 genes in wheat,the Run1 gene in grapevine, and the Rma gene in peanut. Meioticrecombination is essential for classical breeding because it enables thetransfer of favorable alleles across genetic backgrounds, the removal ofdeleterious genomic fragments, and pyramiding traits that aregenetically tightly linked. Therefore, in the absence of accuratemarkers, suppressed recombination forces breeders to enlarge segregatingpopulations for progeny screens.

Phenotypic evaluation of large populations is time-consuming,resource-intensive and not reproducible in every environment.Marker-assisted selection (MAS) offers a feasible alternative. Molecularassays designed to detect unique polymorphisms, such as SNPs, areversatile. However, they may fail to discriminate alleles within andamong lettuce species in a single assay. Structural rearrangements ofchromosomes such as deletions impair hybridization and extension ofsynthetically labeled oligonucleotides. In the case of duplicationevents, multiple copies are amplified in a single reaction withoutdistinction. The development and validation of accurate and highlypredictive markers are therefore essential for successful MAS breedingprograms.

V. Molecular Assisted Breeding Techniques

Genetic markers that can be used in the practice of the presentinvention include, but are not limited to, restriction fragment lengthpolymorphisms (RFLPs), amplified fragment length polymorphisms (AFLPs),simple sequence repeats (SSRs), simple sequence length polymorphisms(SSLPs), single nucleotide polymorphisms (SNPs), insertion/deletionpolymorphisms (Indels), variable number tandem repeats (VNTRs), andrandom amplified polymorphic DNA (RAPD), isozymes, and other markersknown to those skilled in the art. Vegetable breeders use molecularmarkers to interrogate a crop's genome and classify material based ongenetic, rather than phenotypic, differences. Advanced markertechnologies are based on genome sequences, the nucleotide order ofdistinct, polymorphic genotypes within a species. Such platforms enableselection for horticultural traits with markers linked to favorablealleles, in addition to the organization of germplasm using markersrandomly distributed throughout the genome. In the past, a prioriknowledge of the genome lacked for major vegetable crops that now havebeen sequenced. Scientists exploited sequence homology, rather thanknown polymorphisms, to develop marker platforms. Man-made DNA moleculesare used to prime replication of genome fragments when hybridizedpair-wise in the presence of a DNA polymerase enzyme. This synthesis,regulated by thermal cycling conditions that control hybridization andreplication of DNA strands in the polymerase chain reaction (PCR) toamplify DNA fragments of a length dependent on the distance between eachprimer pair. These fragments are then detected as markers and commonlyknown examples include AFLP and RAPD. A third technique, RFLP does notinclude a DNA amplification step and is not discussed here. Amplifiedfragment length polymorphism (AFLP) technology reduces the complexity ofthe genome. First, through digestive enzymes cleaving DNA strands in asequence-specific manner. Fragments are then selected for their size andfinally replicated using selective oligonucleotides, each homologous toa subset of genome fragments. As a result, AFLP technology consistentlyamplifies DNA fragments across genotypes, experiments and laboratories.

In contrast to AFLP, random amplification of polymorphic DNA (RAPD)technology attacks the genome in its full complexity. Typically, asingle primer of 10 nucleotides is used to amplify any fragment of thegenome that, by chance, shows a pattern of tandem homology. It appearsthat the RAPD technology is versatile, low cost and prompt. But whetherit offers a robust marker platform is debated (reviewed in Bardakci,2001). Penner and coworkers (1993) found that RAPD amplification wasinconsistent when comparing results from two oat cultivars (Avena sativaL.) with five primers in seven laboratories. Different size ranges offragments were detected in each laboratory and the majority of fragmentsthat were polymorphic between the cultivars were not amplified in allexperiments. Ghazi et al. (2013) also observed both stable andconflicting bands in separate experiments, despite studying the smallgenome of the prokaryote Streptococcus thermophilus. All authors agreethat minor changes in the experimental conditions of the RAPD techniqueinfluence the amplification and thus the presence of markers. The Centreof Genetic Resources in the Netherlands selectively scores RAPD markerswith a position on the genetic map and a confirmed Mendelian inheritance(www.wageningenur.nl/nl/show/Characteristics-of-genetic-markers-Reproducibility.htm).The USDA(www.download.springer.com/static/pdf/290/chp%253A10.1007%252F978-0-387-30443-4_3.pdf?auth66=1400332645_81ff70ea87777fbaeca0d6d9d1700604&zext=.pdf)argues in favor of elongating RAPD primers to enhance specificity andthereby reproducibility. Nucleotides adjacent to the primers aresequenced and added to primer design and synthesis. Such‘sequence-characterized amplified regions’, or SCAR markers can beapplied to study lettuce genetics. Marker discovery and development incrop plants provides the initial framework for applications tomarker-assisted breeding activities (U.S. Patent Pub. Nos.:2005/0204780, 2005/0216545, 2005/0218305, and 2006/00504538). Theresulting “genetic map” is the representation of the relative positionof characterized loci (polymorphic nucleic acid markers or any otherlocus for which alleles can be identified) to each other.

Polymorphisms comprising as little as a single nucleotide change can beassayed in a number of ways. For example, detection can be made byelectrophoretic techniques including a single strand conformationalpolymorphism (Orita et al. (1989) Genomics, 8(2), 271-278), denaturinggradient gel electrophoresis (Myers (1985) EPO 0273085), or cleavagefragment length polymorphisms (Life Technologies, Inc., Gathersberg,Md.), but the widespread availability of DNA sequencing often makes iteasier to simply sequence amplified products directly. Once thepolymorphic sequence difference is known, rapid assays can be designedfor progeny testing, typically involving some version of PCRamplification of specific alleles (PASA; Sommer, et al. (1992)Biotechniques 12(1), 82-87), or PCR amplification of multiple specificalleles (PAMSA; Dutton and Sommer (1991) Biotechniques, 11(6),700-7002).

Polymorphic markers serve as useful tools for assaying plants fordetermining the degree of identity of lines or varieties (U.S. Pat. No.6,207,367). These markers form the basis for determining associationswith phenotypes and can be used to drive genetic gain. In certainembodiments of methods of the invention, polymorphic nucleic acids canbe used to detect in a lettuce plant a genotype associated with diseaseresistance, identify a lettuce plant with a genotype associated withdisease resistance, and to select a lettuce plant with a genotypeassociated with disease resistance. In certain embodiments of methods ofthe invention, polymorphic nucleic acids can be used to produce alettuce plant that comprises in its genome an introgressed locusassociated with disease resistance. In certain embodiments of theinvention, polymorphic nucleic acids can be used to breed progenylettuce plants comprising a locus associated with disease resistance.

Genetic markers may include “dominant” or “codominant” markers.“Codominant” markers reveal the presence of two or more alleles (two perdiploid individual). “Dominant” markers reveal the presence of only asingle allele. Markers are preferably inherited in codominant fashion sothat the presence of both alleles at a diploid locus, or multiplealleles in triploid or tetraploid loci, are readily detectable, and theyare free of environmental variation, i.e., their heritability is 1. Amarker genotype typically comprises two marker alleles at each locus ina diploid organism. The marker allelic composition of each locus can beeither homozygous or heterozygous. Homozygosity is a condition whereboth alleles at a locus are characterized by the same nucleotidesequence. Heterozygosity refers to different conditions of the allele ata locus.

Nucleic acid-based analyses for determining the presence or absence ofthe genetic polymorphism (i.e. for genotyping) can be used in breedingprograms for identification, selection, introgression, and the like. Awide variety of genetic markers for the analysis of geneticpolymorphisms are available and known to those of skill in the art. Theanalysis may be used to select for genes, portions of genes, QTL,alleles, or genomic regions that comprise or are linked to a geneticmarker that is linked to or associated with disease resistance inlettuce plants.

As used herein, nucleic acid analysis methods include, but are notlimited to, PCR-based detection methods (for example, TaqMan assays),microarray methods, mass spectrometry-based methods and/or nucleic acidsequencing methods, including whole genome sequencing. In certainembodiments, the detection of polymorphic sites in a sample of DNA, RNA,or cDNA may be facilitated through the use of nucleic acid amplificationmethods. Such methods specifically increase the concentration ofpolynucleotides that span the polymorphic site, or include that site andsequences located either distal or proximal to it. Such amplifiedmolecules can be readily detected by gel electrophoresis, fluorescencedetection methods, or other means.

One method of achieving such amplification employs the polymerase chainreaction (PCR) (Mullis et al. 1986 Cold Spring Harbor Symp. Quant. Biol.51:263-273; European Patent 50,424; European Patent 84,796; EuropeanPatent 258,017; European Patent 237,362; European Patent 201,184; U.S.Pat. Nos. 4,683,202; 4,582,788; and 4,683,194), using primer pairs thatare capable of hybridizing to the proximal sequences that define apolymorphism in its double-stranded form. Methods for typing DNA basedon mass spectrometry can also be used. Such methods are disclosed inU.S. Pat. Nos. 6,613,509 and 6,503,710, and references found therein.

Polymorphisms in DNA sequences can be detected or typed by a variety ofeffective methods well known in the art including, but not limited to,those disclosed in U.S. Pat. Nos. 5,468,613, 5,217,863; 5,210,015;5,876,930; 6,030,787; 6,004,744; 6,013,431; 5,595,890; 5,762,876;5,945,283; 5,468,613; 6,090,558; 5,800,944; 5,616,464; 7,312,039;7,238,476; 7,297,485; 7,282,355; 7,270,981 and 7,250,252 all of whichare incorporated herein by reference in their entirety. However, thecompositions and methods of the present invention can be used inconjunction with any polymorphism typing method to type polymorphisms ingenomic DNA samples. These genomic DNA samples used include but are notlimited to, genomic DNA isolated directly from a plant, cloned genomicDNA, or amplified genomic DNA.

For instance, polymorphisms in DNA sequences can be detected byhybridization to allele-specific oligonucleotide (ASO) probes asdisclosed in U.S. Pat. Nos. 5,468,613 and 5,217,863. U.S. Pat. No.5,468,613 discloses allele specific oligonucleotide hybridizations wheresingle or multiple nucleotide variations in nucleic acid sequence can bedetected in nucleic acids by a process in which the sequence containingthe nucleotide variation is amplified, spotted on a membrane and treatedwith a labeled sequence-specific oligonucleotide probe.

Target nucleic acid sequence can also be detected by probe ligationmethods, for example as disclosed in U.S. Pat. No. 5,800,944 wheresequence of interest is amplified and hybridized to probes followed byligation to detect a labeled part of the probe.

Microarrays can also be used for polymorphism detection, whereinoligonucleotide probe sets are assembled in an overlapping fashion torepresent a single sequence such that a difference in the targetsequence at one point would result in partial probe hybridization(Borevitz et al., Genome Res. 13:513-523 (2003); Cui et al.,Bioinformatics 21:3852-3858 (2005). On any one microarray, it isexpected there will be a plurality of target sequences, which mayrepresent genes and/or noncoding regions wherein each target sequence isrepresented by a series of overlapping oligonucleotides, rather than bya single probe. This platform provides for high throughput screening ofa plurality of polymorphisms. Typing of target sequences bymicroarray-based methods is disclosed in U.S. Pat. Nos. 6,799,122;6,913,879; and 6,996,476.

Other methods for detecting SNPs and Indels include single baseextension (SBE) methods. Examples of SBE methods include, but are notlimited, to those disclosed in U.S. Pat. Nos. 6,004,744; 6,013,431;5,595,890; 5,762,876; and 5,945,283.

In another method for detecting polymorphisms, SNPs and Indels can bedetected by methods disclosed in U.S. Pat. Nos. 5,210,015; 5,876,930;and 6,030,787 in which an oligonucleotide probe having a 5′ fluorescentreporter dye and a 3′ quencher dye covalently linked to the 5′ and 3′ends of the probe. When the probe is intact, the proximity of thereporter dye to the quencher dye results in the suppression of thereporter dye fluorescence, e.g. by Forster-type energy transfer. DuringPCR forward and reverse primers hybridize to a specific sequence of thetarget DNA flanking a polymorphism while the hybridization probehybridizes to polymorphism-containing sequence within the amplified PCRproduct. In the subsequent PCR cycle DNA polymerase with 5′→3′exonuclease activity cleaves the probe and separates the reporter dyefrom the quencher dye resulting in increased fluorescence of thereporter.

In another embodiment, a locus or loci of interest can be directlysequenced using nucleic acid sequencing technologies. Methods fornucleic acid sequencing are known in the art and include technologiesprovided by 454 Life Sciences (Branford, Conn.), Agencourt Bioscience(Beverly, Mass.), Applied Biosystems (Foster City, Calif.), LI-CORBiosciences (Lincoln, Nebr.), NimbleGen Systems (Madison, Wis.),Illumina (San Diego, Calif.), and VisiGen Biotechnologies (Houston,Tex.). Such nucleic acid sequencing technologies comprise formats suchas parallel bead arrays, sequencing by ligation, capillaryelectrophoresis, electronic microchips, “biochips,” microarrays,parallel microchips, and single-molecule arrays.

Definitions

The following definitions are provided to better define the presentinvention and to guide those of ordinary skill in the art in thepractice of the present invention. Unless otherwise noted, terms are tobe understood according to conventional usage by those of ordinary skillin the relevant art.

As used herein, the term “plant” includes plant cells, plantprotoplasts, plant cells of tissue culture from which watermelon plantscan be regenerated, plant calli, plant clumps and plant cells that areintact in plants or parts of plants such as pollen, flowers, seeds,leaves, stems, and the like.

As used herein, the term “population” means a genetically heterogeneouscollection of plants that share a common parental derivation.

As used herein, the terms “variety” and “cultivar” mean a group ofsimilar plants that by their genetic pedigrees and performance can beidentified from other varieties within the same species.

As used herein, an “allele” refers to one of two or more alternativeforms of a genomic sequence at a given locus on a chromosome.

A “Quantitative Trait Locus (QTL)” is a chromosomal location thatencodes for at least a first allele that affects the expressivity of aphenotype.

As used herein, a “marker” means a detectable characteristic that can beused to discriminate between organisms. Examples of such characteristicsinclude, but are not limited to, genetic markers, biochemical markers,metabolites, morphological characteristics, and agronomiccharacteristics.

As used herein, the term “phenotype” means the detectablecharacteristics of a cell or organism that can be influenced by geneexpression.

As used herein, the term “genotype” means the specific allelic makeup ofa plant.

As used herein, “elite line” or “cultivated line” means any line thathas resulted from breeding and selection for superior agronomicperformance. An “elite plant” refers to a plant belonging to an eliteline. Numerous elite lines are available and known to those of skill inthe art of lettuce breeding. An “elite population” is an assortment ofelite individuals or lines that can be used to represent the state ofthe art in terms of agronomically superior genotypes of a given cropspecies, such as lettuce. Similarly, an “elite germplasm” or elitestrain of germplasm is an agronomically superior germplasm.

As used herein, the term “introgressed,” when used in reference to agenetic locus, refers to a genetic locus that has been introduced into anew genetic background, such as through backcrossing. Introgression of agenetic locus can be achieved through plant breeding methods and/or bymolecular genetic methods. Such molecular genetic methods include, butare not limited to, various plant transformation techniques and/ormethods that provide for homologous recombination, non-homologousrecombination, site-specific recombination, and/or genomic modificationsthat provide for locus substitution or locus conversion.

As used herein, the term “linked,” when used in the context of nucleicacid markers and/or genomic regions, means that the markers and/orgenomic regions are located on the same linkage group or chromosome suchthat they tend to segregate together at meiosis.

As used herein, “resistance locus” means a locus associated withresistance or tolerance to disease. For instance, a resistance locusaccording to the present invention may, in one embodiment, controlresistance or susceptibility for downy mildew.

As used herein, “resistance allele” means the nucleic acid sequenceassociated with resistance or tolerance to disease.

As used herein “resistance” or “improved resistance” in a plant todisease conditions is an indication that the plant is less affected bydisease conditions with respect to yield, survivability and/or otherrelevant agronomic measures, compared to a less resistant, more“susceptible” plant. Resistance is a relative term, indicating that a“resistant” plant survives and/or produces better yields in diseaseconditions compared to a different (less resistant) plant grown insimilar disease conditions. As used in the art, disease “tolerance” issometimes used interchangeably with disease “resistance.” One of skillwill appreciate that plant resistance to disease conditions varieswidely, and can represent a spectrum of more-resistant or less-resistantphenotypes. However, by simple observation, one of skill can generallydetermine the relative resistance or susceptibility of different plants,plant lines or plant families under disease conditions, and furthermore,will also recognize the phenotypic gradations of “resistant.”

The term “about” is used to indicate that a value includes the standarddeviation of error for the device or method being employed to determinethe value. The use of the term “or” in the claims is used to mean“and/or” unless explicitly indicated to refer to alternatives only orthe alternatives are mutually exclusive, although the disclosuresupports a definition that refers to only alternatives and to “and/or.”When used in conjunction with the word “comprising” or other openlanguage in the claims, the words “a” and “an” denote “one or more,”unless specifically noted. The terms “comprise,” “have” and “include”are open-ended linking verbs. Any forms or tenses of one or more ofthese verbs, such as “comprises,” “comprising,” “has,” “having,”“includes” and “including,” are also open-ended. For example, any methodthat “comprises,” “has” or “includes” one or more steps is not limitedto possessing only those one or more steps and also covers otherunlisted steps. Similarly, any plant that “comprises,” “has” or“includes” one or more traits is not limited to possessing only thoseone or more traits and covers other unlisted traits.

VI. Deposit Information

A deposit was made of at least 2500 seeds of lettuce line CHCG413-0099,which comprises an introgression from L. virosa, as described herein.The deposit was made with the American Type Culture Collection (ATCC),10801 University Boulevard, Manassas, Va. 20110-2209 USA. The deposit isassigned ATCC Accession No. PTA-121598, and the date of deposit was Sep.29, 2014. Access to the deposit will be available during the pendency ofthe application to persons entitled thereto upon request. The depositwill be maintained in the ATCC Depository, which is a public depository,for a period of 30 years, or 5 years after the most recent request, orfor the enforceable life of the patent, whichever is longer, and will bereplaced if nonviable during that period. Applicant does not waive anyinfringement of their rights granted under this patent or any other formof variety protection, including the Plant Variety Protection Act (7U.S.C. 2321 et seq.).

Example 1 Identification of Markers Linked to DM Resistance from L.Virosa

A fragment of the Lactuca virosa genome, introgressed on Chromosome 7 ofL. sativa, was associated with broad resistance to DM and fine-mapped toan interval of 49.8-50.56 cM. This interval appeared to correspond to aphysical size of approximately 1,600 Kb. Subsequently, cultivatedlettuce lines having resistance to DM obtained from L. virosa weresubjected to DNA sequencing using the Sanger method. Results werecompared to cultivated lettuce lines lacking resistance. TheNLSAT009419770 marker (SEQ ID NO: 6) closely linked with DM resistancefrom L. virosa was identified from sequence data, and an assay forplants comprising DM resistance from L. virosa was developed.

This marker was shown to be closely linked to DM resistance from L.virosa, and no breakage between the marker and the resistance has beenobserved. The chromosomal segment containing the marker was confirmed tolocate on chromosome 7.

Example 2 Characterization of an Introgression from L. Virosa onChromosome 7

Microarray DNA fingerprint data from Crisphead coastal lines exhibitingresistance obtained from L. virosa showed an introgression on chromosome7 in an otherwise cultivated background. The identified introgressionwas crossed in the seven different types of lettuce usingmarker-assisted backcrossing (MABC) to test efficacy of theintrogression on Chr. 7. Resulting plants maintained the fullintrogression, and mini-fingerprint data showed that the remaining sizeof the introgression in these plants was as listed in Table 1. All linesdemonstrated resistance, consistent with successful transfer of theintrogression from L. virosa into distinct genetic backgrounds,exclusively using markers. Crisphead lines comprising the introgressionwere tested in the field. Butterhead lines exhibiting resistanceobtained from L. virosa grown in indoor facilities.

TABLE 1 Generation tested and % RP recovered for seven lines comprisingan introgression from L. virosa conferring resistance to DM. Rvir maxRecurrent introgression Parent Market type Generation % RP (cM) CompliceBC3F3 91.4 16.94 Lyra BC3F3 86.5 19.40 Zefira Butterhead, BC3F3 91.922.99 protected culture Freesol Oakleaf, open BC3F3 91.9 17.70 fieldStallion BC2F3 92.9 16.41 PS-6545691 Crisphead BC2F3 89.6 25.76 Coastal,open field RX06413822 Butterhead, BC2F4 93.7 22.99 open field

Example 3 Recombination Screen in Cultivated Lettuce Comprising an L.Virosa Introgression

The physical length of the segment comprising the introgression wasfurther investigated using markers and by sequencing small (200-500 bp)fragments in the region. SNPs were identified within each of the 200-500bp fragments. Using dominant assays, indicative of the absence of elitealleles, it was determined that the introgression started approximatelyat the physical position of ˜53.4 Mb and ended at 56.1 Mb. Physicalpositions refer to the genome sequence of lettuce, which was generatedin a collaboration of the Michelmore lab at UC Davis and the BGI,Shenzen. It was supported by the Lettuce Genome Sequencing Consortium(www.lgr.genomecenter.ucdavis.edu/Home.php).

A recombination screen was conducted in order to identify recombinationevents between three co-dominant markers located within the refinedinterval. In total, 8000 F2 plants were screened for a recombinationevent between the three marker assays in the introgression (FIG. 1).Twenty-two recombinants were identified in the screen and grown in thegreenhouse to produce seed. Seven recombinants, all of which comprised arecombination event between ND0229340 and NLSAT005132975, did not setseed. The fifteen remaining recombinants were capable of producing seed,and were advanced to further linkage drag evaluation in the field aswell as pathology screening to confirm resistance to DM.

Example 4 Pathology Screen of Recombinants

A pathology screen was conducted using 20 seeds of the selfedrecombinants tested with Bremia lactucae race 27. One recombinant eventin line CHCG413-0099, comprising a small introgression of less than0.0125 cM, corresponding to a segment of approximately 250 kb of L.virosa DNA and was still fully resistant to DM.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

1. An elite lettuce plant comprising an introgression from Lactucavirosa on chromosome 7, wherein said introgression comprises a singlegene conferring improved resistance to Bremia lactucae relative to aplant lacking said introgression.
 2. An elite lettuce plant comprisingan introgression from Lactuca virosa on chromosome 7, wherein saidintrogression comprises a first allele conferring improved resistance toBremia lactucae relative to a plant lacking said introgression, andwherein said introgression lacks a second allele genetically linked tosaid first allele and conferring stunted growth.
 3. The elite lettuceplant of claim 2, wherein said introgression from Lactuca virosa islocated between locus NLSAT009419770 (SEQ ID NO: 6) and locusNLSAT005132975 (SEQ ID NO:1) in said plant.
 4. The elite lettuce plantof claim 1, wherein: (a) said introgression from Lactuca virosa is about250 kb or less; (b) the plant comprises a Lactuca virosa allele at locusNLSAT009419770 (SEQ ID NO: 6) and a Lactuca sativa allele at locusNLSAT005132975 (SEQ ID NO:1); (c) the plant comprises a Lactuca virosaallele at locus NLSAT009419770 (SEQ ID NO: 6) and a Lactuca sativaallele at locus ND0229340 (SEQ ID NO: 11); or (d) a sample of seedcomprising said introgression was deposited under ATCC Accession NumberPTA-121598. 5.-7. (canceled)
 8. A plant part of the plant of claim
 1. 9.(canceled)
 10. A method for producing a lettuce plant with improvedresistance to Bremia lactucae, comprising: a) crossing the elite lettuceplant of claim 2 with itself or with a second lettuce plant of adifferent genotype to produce one or more progeny plants; and b)selecting a progeny plant comprising said introgression.
 11. The methodof claim 10, wherein: (a) selecting the progeny plant comprisesidentifying a progeny plant that (1) comprises a Lactuca virosa alleleat a locus genetically linked to said first allele and/or lacks an eliteallele present at the corresponding locus in the elite lettuce plant,and (2) lacks a Lactuca virosa allele at a locus genetically linked tosaid second allele, and/or comprises an allele present at thecorresponding locus from in the elite lettuce plant; (b) the progenyplant is an F2-F6 progeny plant or wherein producing the progeny plantcomprises backcrossing; (c) producing the progeny plant comprisesbackcrossing; or (d) a sample of seed comprising said introgression wasdeposited under ATCC Accession Number PTA-121598.
 12. The method ofclaim 11, wherein selecting a progeny plant comprises (a) detecting atleast one allele at a locus selected from the group consisting ofNLSAT009419770 (SEQ ID NO: 6), ND0229340 (SEQ ID NO: 11) andNLSAT005132975 (SEQ ID NO:1); or (b) detecting an allele at locusNLSAT009419770 and an allele at locus ND0229340.
 13. The method of claim12, wherein (a) the allele(s) are detected by a PCR-based method usingoligonucleotide primer pair(s); and (b) the allele at locusNLSAT009419770 is detected using the primer pair comprising SEQ ID NO: 7and SEQ ID NO: 8; or the allele at locus ND0229340 is detected using theprimer pair comprising SEQ ID NO: 12 and SEQ ID NO: 13; or the allele atlocus NLSAT005132975 is detected using the primer pair comprising SEQ IDNO: 2 and SEQ ID NO:3. 14.-16. (canceled)
 17. The method of claim 11,wherein backcrossing comprises from 2-7 generations of backcrosses. 18.(canceled)
 19. A method for obtaining an elite lettuce plant exhibitingimproved resistance to Bremia lactucae, comprising: a) obtaining anelite lettuce plant heterozygous for a first allele from Lactuca virosathat confers quantitative resistance to Bremia lactucae and that isgenetically linked in the plant to a second allele from Lactuca virosathat confers stunted growth, wherein the plant is heterozygous relativeto a corresponding locus in said elite plant; (b) obtaining progeny ofthe plant; and (c) selecting at least a first progeny plant in whichrecombination has occurred such that the progeny comprises said firstallele that confers quantitative resistance to Bremia lactucae but notsaid second allele.
 20. The method of claim 19, wherein selecting theprogeny plant comprises identifying a progeny plant that (1) comprises aLactuca virosa allele at a locus genetically linked to said first alleleand/or lacks an allele present at the corresponding locus in the elitelettuce plant, and (2) lacks a Lactuca virosa allele at a locusgenetically linked to said second allele, and/or comprises an allelepresent at the corresponding locus from in the elite lettuce plant. 21.The method of claim 20, wherein selecting a progeny plant comprises (a)detecting at least one allele at a locus selected from the groupconsisting of NLSAT009419770 (SEQ ID NO: 6), ND0229340 (SEQ ID NO: 11)and NLSAT005132975 (SEQ ID NO:1); or (b) detecting an allele at locusNLSAT009419770 and an allele at locus ND0229340.
 22. The method of claim21, wherein (a) the allele(s) are detected by a PCR-based method usingoligonucleotide primer pair(s); and (b) the allele at locusNLSAT009419770 is detected using the primer pair comprising SEQ ID NO: 7and SEQ ID NO: 8; or the allele at locus ND0229340 is detected using theprimer pair comprising SEQ ID NO: 12 and SEQ ID NO: 13; or the allele atlocus NLSAT005132975 is detected using the primer pair comprising SEQ IDNO: 2 and SEQ ID NO:3.
 23. (canceled)
 24. A plant or a plant partthereof, produced by the method of claim
 19. 25. (canceled)
 26. Theelite lettuce plant of claim 1, wherein a sample of seed comprising saidintrogression was deposited under ATCC Accession Number PTA-121598. 27.The plant of claim 26, further defined as a plant of lettuce lineCHCG413-0099.
 28. A seed that produces the plant of claim
 26. 29. Amethod for producing a lettuce plant with improved resistance to Bremialactucae, comprising: a) crossing the lettuce plant of claim 26 withitself or with a second lettuce plant of a different genotype to produceone or more progeny plants; and b) selecting a progeny plant comprisingan introgression from Lactuca virosa on chromosome 7, wherein saidintrogression comprises a first allele conferring improved resistance toBremia lactucae relative to a plant lacking said first allele, andwherein said introgression lacks a second allele genetically linked tosaid first allele and conferring stunted growth.
 30. The method of claim29, wherein selecting a progeny plant comprises (a) detecting at leastone allele at a locus selected from the group consisting ofNLSAT009419770 (SEQ ID NO: 6), ND0229340 (SEQ ID NO: 11) andNLSAT005132975 (SEQ ID NO:1); or (b) detecting an allele at locusNLSAT009419770 and an allele at locus ND0229340.
 31. The method of claim30, wherein (a) the allele(s) are detected by a PCR-based method usingoligonucleotide primer pair(s); and (b) the allele at locusNLSAT009419770 is detected using the primer pair comprising SEQ ID NO: 7and SEQ ID NO: 8; or the allele at locus ND0229340 is detected using theprimer pair comprising SEQ ID NO: 12 and SEQ ID NO: 13; or the allele atlocus NLSAT005132975 is detected using the primer pair comprising SEQ IDNO: 2 and SEQ ID NO:3.
 32. (canceled)
 33. A plant produced by the methodof claim 29.